Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance

Kei Ichi Takata, Shelley Reh, Matthew J. Yousefzadeh, Maciej J. Zelazowski, Sarita Bhetawal, David Trono, Megan G. Lowery, Maria Sandoval, Yoko Takata, Yue Lu, Kevin Lin, Jianjun Shen, Donna F. Kusewitt, Kevin M. McBride, Francesca Cole, Richard D. Wood

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3 Scopus citations

Abstract

DNA polymerase ν (pol ν), encoded by the POLN gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν–defective mice and human cells. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln are fertile and have normal testis morphology. However, pol ν–disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ). We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of Poln in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of POLN from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ) supports such a specialized role.

Original languageEnglish (US)
Article numbere1006818
JournalPLoS genetics
Volume13
Issue number6
DOIs
StatePublished - 2017

Bibliographical note

Funding Information:
This research was supported by NIH P01 grant CA193124, the MD Anderson Research Trust and the Grady F. Saunders, PhD Distinguished Research Professorship to RDW and by Cancer Prevention and Research Institute of Texas (CPRIT) R1213 and NIH DP2HD087943 to FC. NIH Grant P30 ES007784 (a pilot project), an Institutional Research Grant (IRG) from The University of Texas MD Anderson Cancer Center, and the Center for Radiation Oncology Research (CROR) supported KT. A CPRIT Core Facility Support Award (RP120348 and RP17002) supported the MD Anderson Molecular Biology Core Facility. The Research Animal Support Facility-Smithville, the Laboratory Animal Genetic Services, the Research Histology, Pathology and Imaging Core, and the Monoclonal Antibody Core, were supported by NIH Cancer Center support grant CA16672 to the University of Texas MD Anderson Cancer Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Marvin Meistrich and Ming Zhao for the experiment separating testicular cells by centrifugal elutriation. We thank Birgitte ?. Wittschieben for establishing 293T-REx POLN cells. We thank Ella Bedford and Pamela Huskey for technical assistance, Nancy Otto and Allegra Abbey for histologic analysis, and Sylvie Doubli? for comments on the manuscript. We are grateful to our laboratory colleagues for discussion. We acknowledge the support of the MD Anderson Molecular Biology Core Facility. We acknowledge the Research Animal Support Facility-Smithville, the Laboratory Animal Genetic Services, the Research Histology, Pathology and Imaging Core, and the Monoclonal Antibody Core.

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